Abstract

Genome wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with atrial fibrillation (AF), but the mechanisms underlying these relationships have not yet been elucidated. Inflammation and fibrosis have been posited as important mechanisms responsible for AF. We sought to investigate the impact of SNP carrier status on inflammation and fibrosis in left atrial appendage tissue.Carrier status of 10 AF-associated SNPs was evaluated on DNA extracted from left atrial appendage tissue in 176 individuals (120 with AF). The presence of inflammation was evaluated through visual quantification of leukocyte infiltration following hematoxylin and eosin staining, while fibrosis was quantified using picrosirius red with fast green staining. Unadjusted and adjusted linear and logistic regression models were utilized to evaluate for an association between SNP carrier status and inflammation and fibrosis. On adjusted logistic regression analysis, the rs7164883 SNP (intronic within HCN4) was associated with a reduced odds of inflammation (odds ratio: 0.42; 95% CI: 0.22-0.81, P = 0.01), and was not associated with fibrosis on adjusted linear regression analysis (β-coefficient: -0.31; 95% CI: -1.03-0.40, P = 0.40). None of the remaining SNPs exhibited significant associations with left atrial inflammation or fibrosis.Among 10 AF-associated SNPs, a single genetic variant was associated with reduced left atrial inflammation, while no histologic differences were observed in the remaining 9. The known AF-associated SNPs do not appear to predispose to the development of pro-inflammatory or pro-fibrotic AF sub-phenotypes.

Abstract

High-burden premature ventricular contractions (PVCs) may be an important determinant of heart failure (HF) in patients presenting for PVC ablation. The prevalence and characteristics of high-burden PVC patients outside this setting remain unknown. We, therefore, sought to determine predictors of high-burden PVCs and, among high-burden PVC patients, predictors of HF.We identified all patients undergoing a 24-48-hour Holter study showing at least 20% PVCs between 2005 and 2013 at the University of California, San Francisco. Three time-matched controls undergoing Holter monitoring were selected for each high-burden PVC patient. Medical records were reviewed and test characteristics of PVC counts from 12-lead electrocardiograms (ECG) as predictors of high-burden PVC Holters were analyzed.Among 5,091 participants, 66 (1.3%) exhibited at least 20% PVCs. After multivariate adjustment, high-burden PVC patients had a three-fold greater odds of HF (odds ratio [OR] 3.15; 95% confidence interval [CI] 1.28-6.50; P = 0.005) and 10-fold greater odds of having a first-degree family member with sudden death (OR 9.97; 95% CI 1.78-60.8; P = 0.011). The C-statistic for the number of PVCs on 12-lead electrocardiogram as a predictor of high-burden PVCs was 0.7949. Among high-burden PVC patients, HF was associated with a history of coronary artery bypass grafting (OR 11.76; 95% CI 1.30-106.49; P = 0.028).Among all undergoing Holter monitoring, 1.3% exhibited high-burden PVCs, a phenomenon associated with HF and a first-degree family history of sudden death. In an analysis restricted to high-burden PVC patients, a history of coronary artery bypass grafting was a predictor of HF.

Abstract

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm-Cpn) assists the in vitro refolding of the well-characterized β-sheet lens protein human γD-crystallin (HγD-Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD-Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm-Cpn suppression of aggregation of these HγD-Crys mutants upon dilution out of denaturant. Mm-Cpn was capable of suppressing the off-pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm-Cpn recognized the HγD-Crys mutants better than (wild-type) WT and refolded most mutant HγD-Crys to levels twice that of WT HγD-Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm-Cpn does not recognize the features of HγD-Crys tested-paired aromatics, exposed domain interface, or destabilized core-but rather recognizes other features of the partially folded β-sheet conformation that are absent or inaccessible in the native state of HγD-Crys.

Abstract

In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.